13-cis-Retinoic acid: inhibition of bladder carcinogenesis induced in rats by N-butyl-N-(4-hydroxybutyl)nitrosamine.

Transitional cell carcinoma was induced in the bladders of male Fischer rats by 12 oral doses of the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine. Feeding of 13-cis-retinoic acid after completion of carcinogen treatment diminished the number and severity of cancers and other proliferative lesions of the bladder.

Mammary carcinogenesis in rats in relation to age at time of N-nitroso-N-methylurea administration.

The effects on mammary carcinogenesis when N-nitroso-N-methylurea (NMU) is administered to rats of different ages were studied. Female outbred Sprague-Dawley rats received two iv injections of NMU (5 mg/100 g body wt/injection) 1 week apart beginning at either 35, 50, 80, 140, or 200 days of age. Animals were killed 6 months after the initial NMU injection, and all mammary tumors were histologically classified. The percent incidence of mammary carcinomas for each age group was as follows: 100%, 35 days old; 94%, 80 days old; 59%, 80 days old; 30%, 140 days old; and 22%, 200 days old. Rats receiving NMU at a young age also exhibited a greater number of carcinomas per rat with latent periods that were in general shorter than those of rats treated at later ages. Since NMU does not require metabolic activation, the observed decrease in chemically induced mammary tumors in aging rats appears to be primarily due to changes occurring within the mammary gland.

Histogenesis and dose dependence of N-methyl-N-nitrosourea-induced carcinoma in a localized area of the hamster trachea.

The effect of the number of weekly intratracheal Instillations of N-methyl-N-nitrosourea (MNU) on induction of tracheal tumors was studied in male noninbred Syrian golden hamsters. The histogenesis of metaplastic and neoplastic lesions was also characterized. Treatment of hamsters once weekly for either 6, 8, 10, 12, or 14 weeks with a 0.5% solution of MNU resulted in the induction of a 0, 6, 11, 26, and 42% incidence of carcinoma, respectively, at 6 months after the first MNU treatment. Of the carcinomas induced, 87% were combined epidermoid and adenocarcinomas, whereas 13% were epidermoid carcinomas. In animals killed 1 week following either 1, 3, 6, 8, 10, 12, or 14 treatments, a continuum of metaplastic and neoplastic changes was observed that correlated well with the cancer incidence exhibited at the termination of the study. Mucous cells were found to be of prime importance in the development of the metaplastic and neoplastic tracheal lesions observed.

Chemoprevention of N-nitroso-N-methylurea-induced mammary cancers by pretreatment with 17 beta-estradiol and progesterone.

Because hormones of pregnancy are thought to alter the mammary gland such that the epithelial cells are less susceptible to future carcinogenic insults, the present study was conducted to determine the ability of short-term treatment with 17 beta-estradiol and/or progesterone, administered immediately after puberty, to prevent mammary cancers in rats subsequently exposed to N-nitroso-N-methylurea [(NMU) CAS:684-93-5]. Beginning at 40 days of age, female outbred Sprague-Dawley rats received 20 micrograms 17 beta-estradiol and/or 4 mg progesterone for 5 weeks. NMU (50 mg/kg body wt) was administered at 96 and 103 days of age (3 and 4 wk, respectively, after the last hormone injection). Pretreatment of rats with 17 beta-estradiol plus progesterone was highly effective in preventing mammary cancer induction (88% fewer cancers compared to the cancer incidence in rats pretreated with the hormone vehicle). Wholemounts of the mammary glands of rats treated with 17 beta-estradiol plus progesterone revealed that the gland was stimulated to a highly differentiated state (similar to that observed in late pregnancy). At the time of NMU treatment, the gland had involuted but was quite different from controls; i.e. an absence of terminal end buds and terminal ducts was noted. The short-term treatment with hormones did not induce tumors and did not interfere with subsequent reproductive and lactational performance. It is apparent that stimulation of the mammary gland to a highly differentiated state early in life can provide protection against future carcinogen exposure.

N-nitroso-N-methylurea-induced mammary carcinogenesis: effect of pregnancy on preneoplastic cells.

The effect of pregnancy and lactation on mammary cancers induced with N-nitroso-N-methylurea (NMU) was determined in female outbred Sprague-Dawley rats. The animals received 5 mg NMU/100 g body weight at 50 days of age and were divided into the following groups: virgin, pregnancy (beginning 10 days after NMU administration), pregnancy and lactation (beginning 10 days after NMU), and pregnancy and lactation (beginning 82 days after NMU). The time of appearance of the first palpable cancers was shorter in rats undergoing an early pregnancy. Few cancers, however, were detected from rats after pregnancy or pregnancy and lactation was completed, and a decrease in cancer incidence from virgin rats was observed in these animals at termination of the study. Since NMU is a direct-acting carcinogen with a short half-life, no effect of pregnancy on carcinogen metabolism or binding could have occurred. Preneoplastic cells present before pregnancy appeared to have been either altered (such that their latent period was increased) or destroyed by the hormones associated with pregnancy.

Carcinogenicity of N-methyl-N-nitrosourea and N-ethyl-N-nitrosourea when applied to a localized area of the hamster trachea.

The carcinogenicity of N-methyl-N-nitrosourea (MNU) and N-ethyl-N-nitrosourea (ENU) was studied with the use of a hamster tracheal tumor model system. Hamsters received 15 or 10 once-weekly treatments of either a 0.5 or 0.25% solution of MNU and were killed 9 months after the first intratracheal instillation. Other hamsters received 15 once-weekly treatments of a 0.5, 0.25, or 0.125% solution of ENU and were killed at 6 months. Treatment with MNU resulted in a dose-dependent induction of tracheal carcinomas; 94% of the tumors induced were combined epidermoid and adenocarcinomas. Treatment of hamsters with a 0.5, 0.25, 0.125% solution of ENU induced an 83, 64, and 71% incidence of benign tracheal tumors, respectively (papillomas and polyps). No tracheal carcinomas were induced by ENU. The carcinogenicity of MNU and the reproducibility of tumor induction with the use of the localized tracheal washing, tumor model system were confirmed. Furthermore, the sensitivity of the localized tracheal washing technique for the detection of the tumorigenicity of compounds toward respiratory epithelium was demonstrated.

Effects of folate deficiency and supplementation on methylnitrosourea-induced rat mammary tumors.

BACKGROUND: There are metabolic and epidemiologic data consistent with the hypothesis that folate deficiency increases the likelihood of cancer. Conversely, it is also known that folate is necessary for cancer growth, but few experiments in laboratory animals have evaluated the effects of folate deficiency on the development of chemically induced cancers. PURPOSE: Our purpose was to determine the effects of nutritional folate deficiency in female Fischer 344 rats on initiation and early promotion of methylnitrosourea (MNU)-induced mammary cancer. METHODS: Rats (age, 27 days) were fed a folic acid-deficient diet (AIN-76A) supplemented with glycine and succinylsulfathiazole [FA(0)]; the FA(0) diet supplemented with 2 or 40 mg of folic acid per kilogram [FA(2) or FA(40), respectively]; or the FA(0) diet supplemented with 20 mg of folinic acid per kilogram [FL(20)]. At 57 days of age, each diet-treated group (30 rats in each group) received MNU (50 mg/kg) by intravenous injection. Immediately after MNU treatment, all animals were fed the AIN-76A complete diet containing 2 mg of folic acid per kilogram. Control groups were fed the AIN-76A complete diet throughout the entire experiment. RESULTS: After 4 weeks, folate deficiency, but not anemia or growth suppression, was documented by lower folate levels in plasma and red blood cells in the group receiving the FA(0) diet. Cancer multiplicity (i.e., number of mammary cancers per number of tumor-bearing animals) at 180 days after MNU injection was 1.32, 1.90, 2.14, and 2.73 mammary cancers per tumor-bearing animal in the FA(0), FA(2), FA(40), and FL(20) groups, respectively; the value in the FA(0) group was statistically significant compared with the values in the other groups. The time required for 50% of the rats to develop palpable mammary cancer was 170, 142, 100, and 85 days, respectively. The value of 170 days for the FA(0) group was statistically significant compared with the values of 100 and 85 days. Mammary cancer incidence was 63%, 70%, 72%, and 73%, respectively; these percentages were not significantly different. CONCLUSIONS: Folate deficiency suppresses and folate supplementation enhances initiation or early promotion of MNU-induced mammary cancer in rats, even when the folate-deficient rats do not have anemia or growth suppression. IMPLICATION: Since the rat is relatively resistant to folate deficiency anemia, other animal models should be used to test the effect of folate nutriture on carcinogenesis.

Inhibition of 7,12-dimethylbenz(a)anthracene-induced mammary carcinogenesis by retinyl acetate.

The administration of 2.5 mg retinyl acetate daily in the diet to female Sprague-Dawley rats beginning 7 days after the intragastric instillation of either 2.5, 5, or 15 mg 7,12-dimethylbenz(a)anthracene (CMBA) resulted in a reduction in the incidence of benign mammary tumors of 37, 30, and 31%, respectively. An equally significant reduction in the number of tumors was also evident. Although no difference was noted in the percentage incidence of mammary adenocarcinomas between the placebo and 2.5 mg retinyl acetate-treated groups at the 2.5-mg DMBA level, the percentage incidence was reduced by 52 and 39% in these groups at the 5- and 15-mg DMBA dose. Furthermore, the number of adenocarcinomas was also significantly reduced. Although both the percentage incidence and number of tumors were reduced by treatment with 1 mg retinyl acetate, these differences were not statistically significant. Liver histology and liver function tests of rats of the retinyl acetate groups did not differ from that of the control group. Similarly, the estrus cycle of treated animals did not differ from that of control rats. These data indicate that relatively large doses of retinyl acetate significantly inhibit the development of DMBA-induced mammary adenocarcinomas and benign tumors. Furthermore, the suppression of mammary tumorigenesis is apparently not the result of an alteration in either the metabolism of DMBA or estrogen nor to an inhibition of tumor growth resulting from retinyl acetate toxicity. The inhibitory effect of retinyl acetate may be related to the effect of retinoids on epithelial cell differentiation and/or reversal of carcinogen-induced anaplasia.

Inhibition of mammary cancer by retinyl methyl ether.

Daily feeding of the synthetic retinoid, retinyl methyl ether, beginning one week after the oral administration of 7,12-dimethylbenz(a)anthracene to female Sprague-Dawley rats, inhibited the incidence of mammary cancer and diminished the number of mammary tumors, both malignant and benign, caused by 7,12-dimethylbenz(a)anthracene. Retinyl methyl ether also markedly increased the latent period for appearance of mammary cancers. Retinyl methyl ether caused no evident toxicity and did not affect weight gain in these experiments. This synthetic retinoid was superior to the natural retinoid, retinyl acetate, for inhibition of mammary carcinogenesis.

Effect of delay in administration of 13-cis-retinoic acid on the inhibition of urinary bladder carcinogenesis in the rat.

The effect of a delay in starting 13-cis-retinoic acid treatment on the inhibition of urinary bladder carcinoma induced by N-butyl-N-(4-hydroxybutyl)nitrosamine was studied in male Fischer 344 rats. Animals received a total p.o. dose of either 1200, 1800 or 2400 mg N-butyl-N-(4-hydroxybutyl)nitrosamine over a period of six weeks. At either one, five, and nine weeks after the last N-butyl-N-(4-hydroxybutyl)nitrosamine intubation, animals were started on a diet supplemented with 13-cis-retinoic acid (240 mg/kg of laboratory chow) or continued on laboratory chow. Animals were killed at one year after the first carcinogen intubation for histological evaluation of the bladder. Feeding of 13-cis-retinoic acid reduced the incidence, average number, and severity of transitional cell carcinomas as well as hyperplasia and cellular atypia. Furthermore, even a nine-week delay in starting the retinoid feeding did not diminish the ability of 13-cis-retinoic acid to inhibit bladder carcinogenesis.

Modulation of biomarkers by chemopreventive agents in smoke-exposed rats.

Chemoprevention opens new perspectives in the prevention of cancer and other chronic degenerative diseases associated with tobacco smoking, exploitable in current smokers and, even more, in exsmokers and passive smokers. Evaluation of biomarkers in animal models is an essential step for the preclinical assessment of efficacy and safety of potential chemopreventive agents. Groups of Sprague Dawley rats were exposed whole body to a mixture of mainstream and sidestream cigarette smoke for 28 consecutive days. Five chemopreventive agents were given either with drinking water (N-acetyl-L-cysteine, 1 g/kg body weight/day) or with the diet (1,2-dithiole-3-thione, 400 mg; Oltipraz, 400 mg; phenethyl isothiocyanate, 500 mg; and 5,6-benzoflavone, 500 mg/kg diet). The monitored biomarkers included: DNA adducts in bronchoalveolar lavage cells, tracheal epithelium, lung and heart; oxidative damage to pulmonary DNA; hemoglobin adducts of 4-aminobiphenyl and benzo(a)pyrene-7,8-diol-9,10-epoxide; micronucleated and polynucleated alveolar macrophages and micronucleated polychromatic erythrocytes in bone marrow. Exposure of rats to smoke resulted in dramatic alterations of all investigated parameters. N-Acetyl-L-cysteine, phenylethyl isothiocyanate, and 5,6-benzoflavone exerted a significant protective effect on all alterations. 1,2-Dithiole-3-thione was a less effective inhibitor and exhibited both a systemic toxicity and genotoxicity in alveolar macrophages, whereas its substituted analogue Oltipraz showed limited protective effects in this model. Interestingly, combination of N-acetyl-L-cysteine with Oltipraz was the most potent treatment, resulting in an additive or more than additive inhibition of smoke-related DNA adducts in the lung and hemoglobin adducts. These results provide evidence for the differential ability of test agents to modulate smoke-related biomarkers in the respiratory tract and other body compartments and highlight the potential advantages in combining chemopreventive agents working with distinctive mechanisms.

Celecoxib inhibits N-butyl-N-(4-hydroxybutyl)-nitrosamine-induced urinary bladder cancers in male B6D2F1 mice and female Fischer-344 rats.

Epidemiological studies have shown that nonsteroidal anti-inflammatory drugs (NSAIDs) may have a role in the prevention of human cancers. A number of preclinical studies have also suggested that inhibition of cyclooxygenase (COX) with NSAIDs has an anticancer effect in animal models of colon, urinary bladder, skin, and breast. In these studies, we evaluated the COX-2 inhibitor celecoxib in two rodent models of urinary bladder cancer. Male B6D2F1 mice treated with N-butyl-N-(4-hydroxybutyl)-nitrosamine (OH-BBN) developed transitional and squamous cell urinary bladder cancers, many of which grew rapidly and caused substantial morbidity that required sacrifice of the mice. Groups of mice received various daily doses of celecoxib in the diet (1250, 500, or 200 mg/kg of diet) beginning 7 days before the initiation of 12 weekly doses of OH-BBN. Mice were checked weekly for the presence of palpable urinary bladder masses. The study was terminated at 8 months following the initial treatment with OH-BBN. The percentage of mice with large palpable bladder lesions, which necessitated sacrifice of the mice, was 40% in the OH-BBN control group. In contrast, only 10% of all celecoxib-treated mice required sacrifice before the scheduled termination of the experiment, implying that all three doses of celecoxib inhibited the formation of large palpable lesions. Celecoxib did not significantly alter the incidence of preneoplastic bladder lesions, but did dose-dependently decrease the total number of urinary bladder cancers/mouse, palpable plus microscopic, by 77, 57, and 43% at dosages of 1250, 500, and 200 mg of celecoxib/kg of diet, respectively. In the second model, female Fischer-344 rats were administered OH-BBN twice/week for a period of 8 weeks. After 8 months, all rats developed preneoplastic lesions, whereas roughly 60% of the rats developed relatively small urinary bladder cancers. Rats were treated continually with celecoxib in the diet (500 or 1000 mg/kg of diet) beginning either 1 week prior to the initial OH-BBN treatment or beginning 1 week following the last OH-BBN treatment. Neither celecoxib treatment regimen significantly altered the number of preneoplastic lesions. Whereas celecoxib treatment initiated prior to OH-BBN administration decreased cancer incidence roughly 65%, celecoxib treatment initiated beginning 1 week after the last dose of OH-BBN profoundly decreased cancer incidence (>95%). Celecoxib did not alter the body weights of the mice or rats, or cause other signs of toxicity at any of the doses studied. Taken together these results demonstrate that: (a) celecoxib effectively inhibits tumor growth and enhances survival in the mouse model of urinary bladder cancer; and (b) celecoxib profoundly inhibits development of urinary bladder cancers in the rat model even when administered following the last dose of OH-BBN. Clinical trials will be necessary to determine whether COX-2 inhibitors will provide a clinical benefit in human bladder cancer.

Patterns of DNA adduct formation in liver and mammary epithelial cells of rats treated with 7,12-dimethylbenz(a)anthracene, and selective effects of chemopreventive agents.

7,12-Dimethylbenz(a)anthracene (DMBA) is a prototype carcinogen that induces a high yield of mammary tumors in rats after a single feeding. We investigated the induction and chemoprevention of DNA adducts in female Sprague Dawley rats receiving DMBA by gavage according to a variety of treatment schedules. The patterns of 32P-postlabeled DNA adducts in liver and mammary epithelial cells were similar to those produced by the in vitro reaction of metabolically activated DMBA with calf thymus DNA. There was a high and statistically significant correlation between dose of DMBA administered to rats (0, 0.6, 2.4, and 12 mg/kg body weight) and levels of DNA adducts in both types of cells. The regression lines relating DMBA doses to total DNA adduct levels were significantly divergent and crossed at 1.5 mg/kg body weight, indicating that, at lower doses, the formation of DNA adducts is more intense in target mammary cells, whereas at higher doses, DNA adduct levels are more elevated in liver cells, presumably due to the greater metabolic capacity of this organ. When the rats were sacrificed 7 days rather than 2 days after DMBA administration, DNA adduct levels were approximately halved in both liver and mammary cells. The observed patterns can be interpreted based on toxicokinetic factors, local and distant metabolism, removal of DNA adducts by excision repair, and cell proliferation rate. Of three chemopreventive agents given with the diet to rats treated with 12 mg of DMBA, 5,6-benzoflavone (1650 ppm) was the most effective, inhibiting DNA adduct formation in liver and mammary cells by 96.5 and 83.5%, respectively. Feeding of 1,2-dithiole-3-thione (600 ppm) inhibited this biomarker by 68.5 and 50.2%, whereas butyl hydroxyanisole (BHA; 5000 ppm) showed a significant inhibition in the liver (46.5%) but was ineffective in mammary cells (29.0%, not significant). These data correlate nicely with the results of a parallel study in which 5,6-benzoflavone, 1,2-dithiole-3-thione, and BHA inhibited formation of hemoglobin adducts by 80.0, 44.0, and 0%, respectively; the incidence of mammary tumors by 82.4, 47.1, and 5.9%, respectively; and their multiplicity by 92.6, 80.0, and 7.4%, respectively. Therefore, biomarkers of biologically effective dose are highly predictive of the efficacy of chemopreventive agents in the DMBA rat mammary model. The selective inhibition by BHA of DNA adducts in the liver but not in mammary cells is consistent with the finding that this phenolic antioxidant stimulated phase II activities in the liver but not in the mammary gland (L. L. Song et al., manuscript in preparation). In any case, the broad-spectrum inducer 5,6-BF appears to be more effective than the two monofunctional phase II inducers, presumably because an enhanced activation of DMBA to reactive metabolites is coordinated with their blocking, detoxification, and excretion.

Inhibition of urinary bladder cancer by N-(ethyl)-all-trans-retinamide and N-(2-hydroxyethyl)-all-trans-retinamide in rats and mice.

The chemopreventive activity of two synthetic retinamides of relatively low toxicity against N-butyl-N-(4-hydroxybutyl)nitrosamine (OH-BBN)-induced urinary bladder cancer was studied in F344 rats and C57BL/6 X DBA/2 F1 mice. Female and male rats were given a total dose of either 1800 or 3200 mg OH-BBN over a period of 6 or 8 weeks, respectively. Male mice were given a total dose of either 90 or 180 mg OH-BBN over a period of 9 weeks. Seven days after the final intubation of a period of 9 weeks. Seven days after the final intubation of OH-BBN, animals were fed either a placebo diet or a diet supplemented with the following retinoids: for rats, 0.8 mmol 13-cis-retinoic acid, 2 mmol N-(ethyl)-all-trans-retinamide, or 2 mmol N-(2-hydroxyethyl)-all-trans-retinamide per kg diet; and for mice, either 0.5 or 1.0 mmol of N-(ethyl)-all-trans-retinamide or N-(2-hydroxyethyl)-all-trans-retinamide per kg diet. Animals were killed 6 months after the initial gastric intubation. In comparison to male and female rats fed placebo diets, all three retinoids reduced the incidence, number, and severity of the low-grade papillary transitional cell carcinomas of the urinary bladder. Similarly, treatment of mice with either of the two retinamides reduced the incidence of highly invasive urinary bladder carcinomas. The chemopreventive effect of the less toxic retinamides was equal to or greater than that of 13-cis-retinoic acid.

Inhibitory effect of 13-cis-retinoic acid on urinary bladder carcinogenesis induced in C57BL/6 mice by N-butyl-N-(4-hydroxybutyl)-nitrosamine.

The effect of 13-cis-retinoic acid on the induction of urinary bladder carcinoma by N-butyl-N-(4-hydroxybutyl)nitrosamine (OH-BBN) was studied in male C57BL/6 mice. Animals received a total dose of either 90 or 140 mg of OH-BBN via gastric intubations of 7.5 or 10.0 mg of OH-BBN 2 times each week for 6 or 7 weeks, respectively. Seven days after the last OH-BBN intubation, animals were fed laboratory chow diet supplemented with either 200 mg of 13-cis-retinoic acid per kg or its placebo. Animals were killed at 6 months after the first carcinogen intubation. Highly invasive squamous and transitional cell carcinomas of the urothelium were found at autopsy. In the majority of these carcinomas, invasion of the bladder muscle wall by tumor cells had occurred. At the two dose levels of OH-BBN, feeding of 13-cis-retinoic acid reduced the incidence of both carcinomas and noninvasive papillomas, as well as the extent of neoplastic development in the urinary bladder. In mice receiving the lower dose of OH-BBN, the feeding of 13-cis-retinoic acid prevented the appearance of both squamous and transitional cell carcinomas with a reduction in incidence from 33 to 0% (p less than 0.01). The results of this study indicate that 13-cis-retinoic acid reduced not only the severity of highly invasive urinary bladder carcinomas but also the incidence of such cancers.

Exisulind, a novel proapoptotic drug, inhibits rat urinary bladder tumorigenesis.

Exisulind (Aptosyn) is a novel antineoplastic drug being developed for the prevention and treatment of precancerous and malignant diseases. In colon tumor cells, the drug induces apoptosis by a mechanism involving cyclic GMP (cGMP) phosphodiesterase inhibition, sustained elevation of cGMP, and protein kinase G activation. We studied the effect of exisulind on bladder tumorigenesis induced in rats by the carcinogen, N-butyl-N-(4-hydroxybutyl) nitrosamine. Exisulind at doses of 800, 1000, and 1200 mg/kg (diet) inhibited tumor multiplicity by 36, 47, and 64% and tumor incidence by 31, 38, and 61%, respectively. Experiments on the human bladder tumor cell line, HT1376, showed that exisulind inhibited growth with a GI(50) of 118 microM, suggesting that the antineoplastic activity of the drug in vivo involved a direct effect on neoplastic urothelium. Exisulind also induced apoptosis as determined by DNA fragmentation, caspase activation, and morphology. Analysis of phosphodiesterase (PDE) isozymes in HT1376 cells showed PDE5 and PDE4 isozymes that were inhibited by exisulind with IC(50)s of 112 and 116 microM, respectively. Inhibition of PDE5 appears to be pharmacologically relevant, because treatment of HT1376 cells increased cGMP and activated protein kinase G at doses that induce apoptosis, whereas cyclic AMP levels were not changed. Immunocytochemistry showed that PDE5 was localized in discrete perinuclear foci in HT1376 cells. Immunohistochemistry showed that PDE5 was overexpressed in human squamous and transitional cell carcinomas compared with normal urothelium. The data lead us to conclude that future clinical trials of exisulind for human bladder cancer treatment and/or prevention should be considered and suggest a mechanism of action involving cGMP-mediated apoptosis induction.

Modulation of methylnitrosourea-induced breast cancer in Sprague Dawley rats by dehydroepiandrosterone: dose-dependent inhibition, effects of limited exposure, effects on peroxisomal enzymes, and lack of effects on levels of Ha-Ras mutations.

Dehydroepiandrosterone (DHEA), the major steroid precursor of androgens and estrogens produced in peripheral tissues in primates, is an effective chemopreventive agent in the N-methyl-N-nitrosourea (MNU)-induced rat mammary tumor model. Dietary DHEA (5-600 ppm; 600 mg/kg diet) was administered beginning 1 week before MNU and administered continually throughout the duration of the experiment. The highest dose of DHEA (600 ppm) significantly decreased tumor incidence from 95 to 45% and increased tumor latency and decreased tumor multiplicity from 4.1 to 0.5 tumors/rat. Lower doses of DHEA (5, 24, and 120 ppm) were also effective, decreasing tumor multiplicity by 28, 40, and 55%, respectively, increasing tumor latency in a dose-dependent manner but only minimally affecting final tumor incidence. DHEA in the diet caused a dose-dependent increase in serum levels of DHEA. The 120-ppm dietary dose of DHEA resulted in serum levels of DHEA of approximately 42 pmol/ml levels, similar to those seen in young humans. When we examined whole mounts of mammary glands derived from rats exposed to higher levels of DHEA (600 ppm), we observed a striking increase in lobular development. The doses of DHEA used in these studies (< or =600 ppm) had minimal effects on the induction of fatty acid CoA synthetase, a peroxisome-associated enzyme. In contrast, a dose of 2000 ppm substantially increased levels of peroxisome-associated fatty acid CoA synthetase. The varied and striking efficacy of DHEA was achieved in the absence of any significant effect on body weight gain in the treated rats. Furthermore, tumors from rats treated with MNU alone or rats treated with MNU plus DHEA were examined for the presence of mutations in the Ha-Ras oncogene. There was a slight decrease in the percentage of tumors bearing Ha-Ras mutations in tumors derived from MNU-control rats as contrasted with tumors from MNU-DHEA (120 and 600 ppm)-treated rats. Based on the striking chemopreventive efficacy of continual exposure to DHEA, we examined the effects of more limited exposure to DHEA. Rats were treated with DHEA for a period of 7 weeks immediately before and after MNU injection. Rats were then placed on the control diet for the ensuing 15 weeks. Even this limited exposure to DHEA for a period of 7 weeks profoundly decreased final tumor incidence and multiplicity. Additionally, we examined the effects of intermittent dosing with DHEA. Rats were treated alternatively at 3-week intervals either with diet containing DHEA or with control diet. It was found that this intermittent dosing with DHEA also substantially inhibited the formation of mammary tumors.

N-(4-Hydroxyphenyl)retinamide, a new retinoid for prevention of breast cancer in the rat.

The synethesis of a new retinoid, N-(4-hydroxyphenyl)-all-trans-retinamide, which has useful biological properties, is described. This retinoid was more potent than retinyl acetate in reversing keratinization caused by retinoid deficiency in tracheal organ culture. It was markedly less toxic than retinyl acetate when fed p.o. to rats over 2-week or 6-month periods. It was an effective agent for inhibition of the development of breast cancer induced in rats by N-nitroso-N-methylurea, although it was not as potent as retinyl acetate in this regard. However, the lesser toxicity of 4-hydroxyphenylretinamide makes it a superior agent for prevention of breast cancer. High-pressure liquid chromatographic analyses of liver and breast extracts from rats treated for 6 months with retinoids show the pharmacokinetic basis for the superiority of 4-hydroxyphenylretinamide; this retinoid and its metabolites were found in high concentrations in breast tissue, without any measurable accumulation in the liver or evident liver toxicity. In contrast, chronic feeding of retinyl acetate caused marked deposition of retinyl esters in the liver and severe hepatotoxicity. Whole mounts of rat mammary glands, made after chronic feeding of 4-hydroxyphenylretinamide, showed that it had a marked antiproliferative effect on mammary epithelium.

Altered gene expression profile in chemically induced rat mammary adenocarcinomas and its modulation by an aromatase inhibitor.

In the present study, competitive cDNA library screening (CCLS) and cDNA microarray analyses were employed to identify differentially expressed genes in methylnitrosourea-induced rat mammary adenocarcinomas. The preliminary screening of 100 000 plaques by CCLS identified 1217 clones with differential expression. Dot-blot analysis of the isolated clones verified differential expression in 471 distinct genes. Confirmation of these 471 genes was conducted by performing reverse transcription-polymerase chain reactions, and a total of 160 genes were confirmed after comparing six rat mammary adenocarcinomas and three normal rat mammary glands. Fifty-nine of these showed lower expression in the adenocarcinomas while the remaining 101 were overexpressed in the tumors. Employing a cDNA microarray containing 588 known genes revealed an additional 33 differentially expressed genes in these tumors. Importantly, most of the identified genes demonstrated relatively reproducible overexpression or underexpression in individual tumors. Many of the altered genes determined by cDNA microarray analysis were oncogenes, tumor suppressor genes, or genes involved in cell cycle control and apoptosis. CCLS identified many others not previously associated with mammary carcinogenesis, including a novel gene named RMT-7. Preliminary studies to determine the applicability of this gene expression approach for detecting potential biomarkers for cancer chemoprevention was evaluated in rat mammary tumors obtained from animals treated with vorozole, a potent aromatase inhibitor. When genes exhibiting differential expression as determined by CCLS or cDNA microarray analysis were examined in control and vorozole-treated tumors, expression of 19 genes was found to be modulated significantly in tumors treated with vorozole. Further investigations into these identified genes should contribute significantly to our understanding of the molecular mechanisms of rat mammary tumorigenesis. In addition, the identified genes may become useful targets for drug development and potential biomarkers for monitoring treatment and prevention of breast cancer in humans.

Effects of acute and chronic body weight gain reductions in the evaluation of agents for efficacy in mammary cancer prevention.

Studies were performed to determine the effects of moderate decreases in body weight gain on mammary carcinogenesis. The levels of depressions in weight gain were those often observed in the evaluation of chemopreventive agents. In the first experiment, the effects of acute and chronic reductions of body weight gain when started after carcinogen treatment were examined in young rats (MNU at 50 days of age). Significant decreases (36%) in mammary cancers occurred in groups of rats that underwent a 12% acute reduction in body weight gain as compared with ad libitum controls. In contrast, chronic weight reductions of up to 12% had minimal effects on cancer multiplicities, while a 15% chronic reduction significantly decreased cancer numbers (26%). A second experiment evaluated the efficacy of toremifene (7.0 mg/kg diet), an estrogen/anti-estrogen, and the effect of toremifene-matched body weight gain reduction that occurred during the study. Toremifene caused a chronic reduction in body weight that resulted in a 10% decrease in final body weight at the end of the study. While toremifene-treated rats exhibited a 67% decrease in the number of mammary cancers, the rats which similarly exhibited a 10% decrease in final body weight showed only a 14% decrease in cancer number. Thus, the weight effects observed with toremifene, similar estrogens/anti-estrogens, and other classes of chemopreventive compounds (where chronic body weight reductions are 10% or less) imply that the body weight reduction has a limited effect on overall chemopreventive activity. A third study examined the effect of chronic body weight gain reduction on mammary cancers induced in older rats (MNU given at 100 days of age). This model more closely resembles the status of the breast tissue of mature women currently enrolled in clinical trials of chemopreventive agents. Under these conditions chronic reductions in body weight up to 15% had minimal effects on mammary carcinogenesis. These data further demonstrated that acute body weight reductions in young rats at the time of carcinogen treatment can be a concern in interpretation of the chemopreventive activity of an agent, but that moderate chronic depressions of body weight gain probably do not play a significant role.